The intestinal mucosa exists in a functional equilibrium with the complex lumenal milieu, which is dominated by a wide variety of commensal microbial species. Despite the continuous confrontation with bacterial products the normal mucosa exists in a state of minimal immune activation but must promptly mount an effective defense typically dependent on activation of inflammatory pathway when infection with a pathogen occurs. The overall goal of the present proposal is to define the mechanisms through which the epithelium interfaces with normal and pathogenic lumenal bacteria and the processes determining the functional outcome of those interactions whether normal homeostasis or active inflammation. These studies are based on the hypothesis that a recently identified family of pattern recognition receptor designated toll-like receptors (TLRs) found in preliminary studies to be present on the apical surface of intestinal epithelial cells allow the mucosa to sample the bacterial milieu and play a determining role in the functional outcome of that interaction. We also hypothesize that functional dysregulation of the TLR mediated responses eventuate in chronic intestinal inflammation. These preliminary studies have suggested that TLR4 which binds the ubiquitous bacterial cell wall product lipopolysaccharide (LPS) and TLR3 which binds other cell wall products may be especially pivotal as suggested by the finding of distinctive alterations in expression in human inflammatory bowel disease. These hypotheses will be evaluated through studies designed to address three specific aims: (I) To define the signaling pathways activated in intestinal epithelial cells by different TLRs following binding of cognate ligands, (II) To delineate the functional response of intestinal epithelial cells mediated through activation of TLRs, and (III) To determine the expression and functions of TLRs in inflammatory bowel disease in murine models and human IBD. These studies will encompass detailed assessment of the function of TLRs in model intestinal epithelial cells in vitro and both human tissues and novel murine models in vivo. Collectively these studies should provide new insights into the role of the TLRs, key elements of the innate immune system, in modulating the dynamic balance between controlled surveillance and appropriate response to mucosal challenge by lumenal flora.